Mechanisms of enhanced thrombus formation in cerebral microvessels of mice expressing hemoglobin-S

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264

AbstractFor a long time, blood coagulation and innate immunity have been viewed as interrelated responses. Recently, the presence of leukocytes at the sites of vessel injury has been described. Here we analyzed interaction of neutrophils, monocytes, and platelets in thrombus formation after a laser-induced injury in vivo. Neutrophils immediately adhered to injured vessels, preceding platelets, by binding to the activated endothelium via leukocyte function antigen-1–ICAM-1 interactions. Monocytes rolled on a thrombus 3 to 5 minutes postinjury. The kinetics of thrombus formation and fibrin generation were drastically reduced in low tissue factor (TF) mice whereas the absence of factor XII had no effect. In vitro, TF was detected in neutrophils. In vivo, the inhibition of neutrophil binding to the vessel wall reduced the presence of TF and diminished the generation of fibrin and platelet accumulation. Injection of wild-type neutrophils into low TF mice partially restored the activation of the blood coagulation cascade and accumulation of platelets. Our results show that the interaction of neutrophils with endothelial cells is a critical step preceding platelet accumulation for initiating arterial thrombosis in injured vessels. Targeting neutrophils interacting with endothelial cells may constitute an efficient strategy to reduce thrombosis.

mentioning

confidence: 76%

Tissue factor–positive neutrophils bind to injured endothelial wall and initiate thrombus formation

Darbousset

Thomas

Mezouar

et al. 2012

262

264

“…39 Similarly, the activation of leukocytes, including PMNs, is also important in the formation of a thrombus following cerebral injuries. 40 Thus, the understanding of the mechanisms leading to thrombus formation may help to develop efficient alternative strategies to prevent thrombo-inflammation. Our results indicate that preventing the activation of both PMNs and platelets, by targeting the P2X1 receptor, could potentially represent a therapeutic strategy to prevent arterial thrombosis.…”

Section: Discussionmentioning

confidence: 99%

P2X1 expressed on polymorphonuclear neutrophils and platelets is required for thrombosis in mice

Darbousset

Delierneux

Mezouar

et al. 2014

Key Points• Activation of PMNs at the site of injury is required for thrombin generation.• P2X1 receptor expressed on both PMNs and platelets is crucial to initiate thrombosis.Adenosine triphosphate (ATP) and its metabolite, adenosine, are key regulators of polymorphonuclear neutrophil (PMN) functions. PMNs have recently been implicated in the initiation of thrombosis. We investigated the role of ATP and adenosine in PMN activation and recruitment at the site of endothelial injury. Following binding to the injured vessel wall, PMNs are activated and release elastase. The recruitment of PMNs and the subsequent fibrin generation and thrombus formation are strongly affected in mice deficient in the P2X1-ATP receptor and in wild-type (WT) mice treated with CGS 21680, an agonist of the A2A adenosine receptor or NF449, a P2X1 antagonist. Infusion of WT PMNs into P2X1-deficient mice increases fibrin generation but not thrombus formation. Restoration of thrombosis requires infusion of both platelets and PMNs from WT mice. In vitro, ATP activates PMNs, whereas CGS 21680 prevents their binding to activated endothelial cells. These data indicate that adenosine triphosphate (ATP) contributes to polymorphonuclear neutrophil (PMN) activation leading to their adhesion at the site of laser-induced endothelial injury, a necessary step leading to the generation of fibrin, and subsequent platelet-dependent thrombus formation. Altogether, our study identifies previously unknown mechanisms by which ATP and adenosine are key molecules involved in thrombosis by regulating the activation state of PMNs. (Blood. 2014;124(16):2575-2585

“…For platelet-depletion experiments, antiplatelet serum (10 L in PBS IP; Accurate Chemical and Scientific) was applied (supplemental Figure 2). Removal of circulating neutrophils from Tregdepleted DEREG mice was achieved by IP injecting 75 g of an antineutrophil serum (ANS, RB6-8C5; eBiosciences) 14 or control serum (AIS403; Accurate Chemical and Scientific; supplemental Figure 3). …”

Section: Micementioning

confidence: 99%

Regulatory T cells are strong promoters of acute ischemic stroke in mice by inducing dysfunction of the cerebral microvasculature

Kleinschnitz

Kraft

Dreykluft

et al. 2013

296

293

Key Points• Regulatory T cells are promoters of ischemic stroke by inducing dysfunction of the cerebral microvasculature. We have recently identified T cells as important mediators of ischemic brain damage, but the contribution of the different T-cell subsets is unclear. Forkhead box P3 (FoxP3)-positive regulatory T cells (Tregs) are generally regarded as prototypic antiinflammatory cells that maintain immune tolerance and counteract tissue damage in a variety of immune-mediated disorders. In the present study, we examined the role of Tregs after experimental brain ischemia/reperfusion injury. Selective depletion of Tregs in the DEREG mouse model dramatically reduced infarct size and improved neurologic function 24 hours after stroke and this protective effect was preserved at later stages of infarct development. The specificity of this detrimental Treg effect was confirmed by adoptive transfer experiments in wild-type mice and in IntroductionIschemic stroke induces a profound local inflammatory response involving various types of immune cells that transmigrate across the activated blood-brain barrier to invade the brain in a timed fashion. 1 Although previous research mainly focused on the role of innate immune cells, 2 recent evidence suggests that T cells, which belong to the adaptive immune system, also contribute critically to stroke development, especially in the early phase. 3 T cells have been identified in the postischemic brain as soon as 24 hours after reperfusion, 4 and Abs directed against vascular adhesion receptors expressed on the brain endothelium or leukocyte very late antigen-4 (VLA-4) expressed on lymphocytes inhibited T-cell transmigration and reduced tissue damage in models of stroke. 5 We and others showed recently that recombination activating gene (Rag1)-deficient mice, which lack functional T cells, are largely resistant against ischemic neurodegeneration. 6-8 T cellmediated brain damage became manifest by 24 hours after transient middle cerebral artery occlusion (tMCAO) and did not depend on antigen recognition or costimulation. 8 This clearly argues against TCR-driven mechanisms of tissue damage and suggests instead that T cells act detrimentally in ischemic stroke through antigenindependent pathways, at least during the early phase. Moreover, Rag1 Ϫ/Ϫ mice did not display a gross defect in thrombus formation after artificial vessel wall injury, which could easily explain the stroke protective phenotype in these animals. 8 Although the deleterious effects of T cells in stroke pathophysiology are well accepted, the functional relevance of the different T-cell subsets for stroke progression is less clear, as is their pathologic contribution at the different stages of cerebral ischemia (ie, acute versus chronic). Using adoptive cell transfer in Rag1 Ϫ/Ϫ mice, we could demonstrate that natural killer T cells (NKT cells) Submitted April 26, 2012; accepted October 27, 2012.Prepublished online as Blood First Edition paper, November 15, 2012; DOI 10.1182/blood-2012-04-426734. *C.K. and P.K. ...